In the post-genomic era, high through-put genome-wide scans and SNP association studies hold the promise of identifying new genes as significant risk factors for coronary artery disease (CAD) and myocardial infarction (MI). Such information raises a new challenge: how does an identified gene product influence disease? This was the challenge that emerged from GeneQuest I. This large-scale SNP case-control study in patients with familial, premature CAD/MI, identified three genes that exhibited a significant association with MI. All three were members of the thrombospondin (TSP) gene family. The remarkable clustering of genes within a single family strongly implicates the TSP family in cardiovascular pathology, and this link is further bolstered by the replication of the TSP-2 and TSP-4 associations with MI in separate clinical studies and our own preliminary data showing that each of the three TSP SNP set displays distinct functional differences. This project will focus on TSP-4 and TSP-2. In TSP-4, the SNP leads to a A387 P substitution, occurs at high frequency (34% in the Caucasian population), and increases the risk of MI by almost 2-fold. In TSP-2, the SNP is a t3943g substitution in the 3'-untranslated region, occurs with high frequent (10%) and is protective (approximately 3-fold reduction in MI). The hypothesis to be tested is: the SNPs in TSP-4 and TSP-2 alter protein (TSP-4) or mRNA (TSP-2) structure, which affects the function of TSP-4 and the expression of TSP-2. In turn, these alterations affect the responses of vascular cells, creating a pro- or anti-atherogenic environment in the vessel wall. To test this hypothesis, we will: 1) examine the differential effects of the TSP-4 variant proteins at a molecular and cellular level; 2) assess how the TSP-2 SNPs differentially regulate expression at the transcriptional, post-transcriptional or translational levels; 3) use transgenic mice to explore how the TSPs influence development of atherosclerosis and vascular injury; 4) examine normal and atherosclerotic human vessels for SNP-dependent changes in mRNA and protein; 5) determine if the SNP variants induce different gene profiles consistent with their atherogenic effects; and 6) exploit the GeneBank of cardiology patients to verify the athero-protective effect of the TSP-2 SNP. Together, these studies should provide a rigorous test of the hypothesis and extend our knowledge of the roles of the TSPs in cardiovascular biology.